Tripartite_analysis_manuscript

1 Growth Analysis for Moss and Cyanobacteria

1.1 Introduction

The symbiosis between Sphagnum mosses and cyanobacteria has long been recognized (Limpricht, 1890) and has been implicated in improved moss growth. This together with evidence form other plant - cyanobacteria symbioses suggest that this is not just a symbiosis (interacting species), but perhaps a mutualsitc interaction – thereby benefiting both interacting speices. To investigate this further, Colleen Hulsey and Megan Gable conducted a numer of experiments to investigate the growth consequences of cyanobacteria and sphagnum symbiosis on growth patterns at differing pH. conditions.

1.1.1 Growth 2018 symbiosis approach and measurements

*Experimental conditions & design +conducted on 08/09/2018 +plants pH nitrogen depleted for 2 weeks prior to start +experiment ran for 2 weeks +condition 1: Nitrogen (N), 6 replicates -N, 2 replicates for low N (5mL/L NaNO3– Mm?) +Condition 2: pH, 3.5, 5.5, 8.5 +speices: Sphagnum fallax MN alone, Nostoc muscorum 1037 alone, both species together +amounts: 1 moss capitulum, 0.1g cyanobacteria +3 - 12 well plates, 2 ml liquid medium per well.

*Measurements +fluorcam: area_mm2, QY_max (initial, weekly, final) +pH initial, pH weekly, pH final +weight beginning and end after water removed with centrifugation

*tests +simple scatter and boxplots to view realtionship

1.1.2 Growth 2018 data import and summary

library(tidyverse)
#knitr by default will set the working directory to the source file location
#setwd("~/Documents/Manuscripts:data/Symbiosis_study_2018")
dat1 <- read_csv("fallax_cyano_symbiosis_colleen_10_01_2018.csv") %>% mutate(delta_weight = delta_weight_g *1000)
names(dat1)

## [1] "Sample_well"        "component_measured" "condition"         
## [4] "initial_pH"         "final_pH"           "initial_weight_g"  
## [7] "final_weight_g"     "delta_weight_g"     "delta_weight"

summary(dat1)

##  Sample_well        component_measured  condition           initial_pH   
##  Length:72          Length:72          Length:72          Min.   :3.500  
##  Class :character   Class :character   Class :character   1st Qu.:3.500  
##  Mode  :character   Mode  :character   Mode  :character   Median :5.500  
##                                                           Mean   :5.833  
##                                                           3rd Qu.:8.500  
##                                                           Max.   :8.500  
##                                                                          
##     final_pH     initial_weight_g  final_weight_g    delta_weight_g    
##  Min.   :3.700   Min.   :0.00140   Min.   :0.01430   Min.   :-0.03560  
##  1st Qu.:5.800   1st Qu.:0.01342   1st Qu.:0.04083   1st Qu.: 0.01082  
##  Median :6.200   Median :0.09345   Median :0.09570   Median : 0.03030  
##  Mean   :6.117   Mean   :0.06677   Mean   :0.10127   Mean   : 0.03766  
##  3rd Qu.:6.700   3rd Qu.:0.10622   3rd Qu.:0.15368   3rd Qu.: 0.06940  
##  Max.   :7.440   Max.   :0.12040   Max.   :0.22060   Max.   : 0.11090  
##  NA's   :12      NA's   :12        NA's   :12                          
##   delta_weight   
##  Min.   :-35.60  
##  1st Qu.: 10.82  
##  Median : 30.30  
##  Mean   : 37.66  
##  3rd Qu.: 69.40  
##  Max.   :110.90  
## 

dim(dat1)

## [1] 72  9

1.1.3 Growth 2018 data boxplot analysis

1. Delta growth (weight) by organism regardless of pH; 2. Delta growth by organism and by pH; 3. Delta growth for entier wel by pH

Fig. 1: 2018 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone and when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Cyano in symbiosis includes, endophytes, epiphytes and free living cyano. Data incuded all pH conditions

Fig. 2: 2018 data, change in growth among interacting partners faceted by starting pH condiiton. Measured components as described in fig. 1

Fig. 3: 2018 data, total well biomass change from experiment start to finish. Includes moss + cyano endophyte and free living cyanobacteria not attached to plant

## Warning: Removed 12 rows containing non-finite values (stat_boxplot).

## Warning: Removed 12 rows containing missing values (geom_point).

Fig. 4: 2018 data, change in pH change as measured by (final - initial values)

## Warning: Removed 12 rows containing non-finite values (stat_boxplot).

## Warning: Removed 12 rows containing missing values (geom_point).

Fig. 5: 2018 data, absolute value of pH change as measured by (final - initial values)

1.2 Growth 2017 symbiosis approach and measurements

need experimental details here…

1.2.1 Growth 2017 data import and summary

dat2 <- read_csv("fallax_cyano_symbiosis_allyssa_colleen_2017.csv")
dat3 <- dat2 %>% filter(pH == 3.5 | pH == 5.5 | pH == 8.5)
names(dat3)

## [1] "condition"      "replicate"      "pH"             "plate"         
## [5] "well"           "weight_initial" "weight_final"   "delta_weight"

summary(dat3)

##   condition           replicate         pH            plate   
##  Length:72          Min.   :1.0   Min.   :3.500   Min.   :1   
##  Class :character   1st Qu.:2.0   1st Qu.:3.500   1st Qu.:3   
##  Mode  :character   Median :3.5   Median :5.500   Median :5   
##                     Mean   :3.5   Mean   :5.833   Mean   :5   
##                     3rd Qu.:5.0   3rd Qu.:8.500   3rd Qu.:7   
##                     Max.   :6.0   Max.   :8.500   Max.   :9   
##                     NA's   :18                    NA's   :18  
##       well        weight_initial   weight_final     delta_weight    
##  Min.   : 1.000   Min.   : 3.11   Min.   :  8.67   Min.   :-21.330  
##  1st Qu.: 3.000   1st Qu.:21.73   1st Qu.: 29.20   1st Qu.:  8.562  
##  Median : 6.500   Median :30.00   Median : 46.91   Median : 23.870  
##  Mean   : 6.333   Mean   :33.72   Mean   : 64.98   Mean   : 32.358  
##  3rd Qu.: 9.000   3rd Qu.:40.02   3rd Qu.: 96.84   3rd Qu.: 46.220  
##  Max.   :12.000   Max.   :87.22   Max.   :177.43   Max.   :127.830  
##  NA's   :18       NA's   :18      NA's   :18

dim(dat3)

## [1] 72  8

1.2.2 Boxplot analysis of 2017 data

Fig. 6: 2017 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Conditions aggregated over pH

Fig. 7: 2017 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Similar to Fig. 6 except faceted by pH

Fig. 8: Combined 2018 and 2017 data for change in growth (initial weight (wt) - among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Data is faceted by pH

1.3 ANOVA analysis of 2017

Specifically testing if cyano and moss grown alone, increased in growth at a different rate than when grown together in smbiosis.

2017 Sphagnum-Cyano Import

2017 Summary statistics and graphs

Confirms similar info to 2018 data. Cyano & moss grown together have higher weight than grown separate or grown alone. Higher pHs correspond to produce higher growth with the symbiotic relationship increasing growth response as pH increases.

2017 Two-way ANOVA

##              Df Sum Sq Mean Sq F value   Pr(>F)    
## condition     3  20854    6951   9.251 4.05e-05 ***
## pH            2   8810    4405   5.862  0.00473 ** 
## condition:pH  6   2464     411   0.546  0.77075    
## Residuals    60  45086     751                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = delta_weight ~ condition + pH + condition:pH, data = tri.2017)
## 
## $condition
##                                                               diff
## Cyanobacteria_moss_seperate-cyano_alone                   23.38444
## Cyanobacteria_moss_symbiosis-cyano_alone                  45.84889
## moss_alone-cyano_alone                                    11.11278
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate  22.46444
## moss_alone-Cyanobacteria_moss_seperate                   -12.27167
## moss_alone-Cyanobacteria_moss_symbiosis                  -34.73611
##                                                                 lwr
## Cyanobacteria_moss_seperate-cyano_alone                   -0.761391
## Cyanobacteria_moss_symbiosis-cyano_alone                  21.703053
## moss_alone-cyano_alone                                   -13.033058
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate  -1.681391
## moss_alone-Cyanobacteria_moss_seperate                   -36.417502
## moss_alone-Cyanobacteria_moss_symbiosis                  -58.881947
##                                                                upr
## Cyanobacteria_moss_seperate-cyano_alone                   47.53028
## Cyanobacteria_moss_symbiosis-cyano_alone                  69.99472
## moss_alone-cyano_alone                                    35.25861
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate  46.61028
## moss_alone-Cyanobacteria_moss_seperate                    11.87417
## moss_alone-Cyanobacteria_moss_symbiosis                  -10.59028
##                                                              p adj
## Cyanobacteria_moss_seperate-cyano_alone                  0.0610500
## Cyanobacteria_moss_symbiosis-cyano_alone                 0.0000289
## moss_alone-cyano_alone                                   0.6191255
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate 0.0771172
## moss_alone-Cyanobacteria_moss_seperate                   0.5396394
## moss_alone-Cyanobacteria_moss_symbiosis                  0.0018777
## 
## $pH
##                   diff       lwr      upr     p adj
## pH_5.5-pH_3.5 14.10375 -4.913481 33.12098 0.1842030
## pH_8.5-pH_3.5 27.08708  8.069852 46.10431 0.0031737
## pH_8.5-pH_5.5 12.98333 -6.033898 32.00056 0.2367043

2017 Testing ANOVA assumptions

## 
##  Shapiro-Wilk normality test
## 
## data:  tri2017.residuals
## W = 0.96343, p-value = 0.03459

## Loading required package: carData

## 
## Attaching package: 'car'

## The following object is masked from 'package:dplyr':
## 
##     recode

## The following object is masked from 'package:purrr':
## 
##     some

## Levene's Test for Homogeneity of Variance (center = median)
##       Df F value  Pr(>F)  
## group 11  2.0352 0.04026 *
##       60                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

2017 Nonparametric Tests

## Coefficient covariances computed by hccm()

## 
## DV:  delta_weight 
## Observations:  72 
## D:  1 
## MS total:  438

1.3.1 2017 Data Transformations

2017 Normality and Variance correction

## Warning in sqrt(weight): NaNs produced

## 
## Call:
## lm(formula = logvars ~ logmeans)
## 
## Residuals:
##          1          2          3 
## -0.0006837  0.0018373 -0.0011536 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)   
## (Intercept) 0.698544   0.012165   57.42  0.01109 * 
## logmeans    1.508940   0.008162  184.87  0.00344 **
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.002275 on 1 degrees of freedom
## Multiple R-squared:      1,  Adjusted R-squared:  0.9999 
## F-statistic: 3.418e+04 on 1 and 1 DF,  p-value: 0.003444

## [1] 0.24553

2017 Testing ANOVA - Square root

##              Df Sum Sq Mean Sq F value   Pr(>F)    
## condition     3  95.63   31.88   7.630 0.000262 ***
## pH            2  55.15   27.58   6.601 0.002823 ** 
## condition:pH  6  22.00    3.67   0.878 0.517890    
## Residuals    51 213.08    4.18                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 9 observations deleted due to missingness

## 
##  Shapiro-Wilk normality test
## 
## data:  tri2017.transresiduals
## W = 0.98782, p-value = 0.7902

## Levene's Test for Homogeneity of Variance (center = median)
##       Df F value  Pr(>F)  
## group 11   2.609 0.01021 *
##       51                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Testing ANOVA - Exp Transform

##              Df Sum Sq Mean Sq F value   Pr(>F)    
## condition     3  4.334  1.4447   8.339 0.000131 ***
## pH            2  2.228  1.1139   6.429 0.003236 ** 
## condition:pH  6  0.897  0.1496   0.863 0.528150    
## Residuals    51  8.836  0.1733                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 9 observations deleted due to missingness

## 
##  Shapiro-Wilk normality test
## 
## data:  tri2017.transresiduals2
## W = 0.98278, p-value = 0.5233

## Levene's Test for Homogeneity of Variance (center = median)
##       Df F value  Pr(>F)  
## group 11  2.1987 0.02899 *
##       51                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

1.4 ANOVA analysis of 2018

2018 Spagnum_Cyano Import

## 'data.frame':    48 obs. of  3 variables:
##  $ pH          : num  3.5 3.5 3.5 3.5 5.5 5.5 5.5 5.5 8.5 8.5 ...
##  $ condition   : Factor w/ 6 levels "cyano_alone",..: 5 5 5 5 5 5 5 5 5 5 ...
##  $ delta_weight: num  10.3 11 32 77.3 19.3 23.6 49.2 50 45.5 28.2 ...

## 'data.frame':    48 obs. of  3 variables:
##  $ pH          : Factor w/ 3 levels "pH_3.5","pH_5.5",..: 1 1 1 1 2 2 2 2 3 3 ...
##  $ condition   : Factor w/ 4 levels "moss_alone","cyano_alone",..: 1 1 1 1 1 1 1 1 1 1 ...
##  $ delta_weight: num  10.3 11 32 77.3 19.3 23.6 49.2 50 45.5 28.2 ...

##         
##          moss_alone cyano_alone Cyanobacteria_moss_symbiosis
##   pH_3.5          4           4                            4
##   pH_5.5          4           4                            4
##   pH_8.5          4           4                            4
##         
##          Cyanobacteria_moss_seperate
##   pH_3.5                           4
##   pH_5.5                           4
##   pH_8.5                           4

2018 Summary statistics and graphs

Data suggests that growth is driven by both condition and pH and that condition influences responses to pH. Graphs show support for the idea that at higher pH there is increased growth of Sphagnum, indicative of a symbiotic relationship.

2018 Two-way ANOVA

##              Df Sum Sq Mean Sq F value   Pr(>F)    
## condition     3  11694    3898   5.216  0.00429 ** 
## pH            2  32248   16124  21.577 6.93e-07 ***
## condition:pH  6  11846    1974   2.642  0.03159 *  
## Residuals    36  26902     747                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = delta_weight ~ condition + pH + condition:pH, data = tri.2018)
## 
## $condition
##                                                                diff
## cyano_alone-moss_alone                                   -11.858333
## Cyanobacteria_moss_symbiosis-moss_alone                   26.866667
## Cyanobacteria_moss_seperate-moss_alone                    20.800000
## Cyanobacteria_moss_symbiosis-cyano_alone                  38.725000
## Cyanobacteria_moss_seperate-cyano_alone                   32.658333
## Cyanobacteria_moss_seperate-Cyanobacteria_moss_symbiosis  -6.066667
##                                                                 lwr
## cyano_alone-moss_alone                                   -41.915110
## Cyanobacteria_moss_symbiosis-moss_alone                   -3.190110
## Cyanobacteria_moss_seperate-moss_alone                    -9.256777
## Cyanobacteria_moss_symbiosis-cyano_alone                   8.668223
## Cyanobacteria_moss_seperate-cyano_alone                    2.601556
## Cyanobacteria_moss_seperate-Cyanobacteria_moss_symbiosis -36.123444
##                                                               upr
## cyano_alone-moss_alone                                   18.19844
## Cyanobacteria_moss_symbiosis-moss_alone                  56.92344
## Cyanobacteria_moss_seperate-moss_alone                   50.85678
## Cyanobacteria_moss_symbiosis-cyano_alone                 68.78178
## Cyanobacteria_moss_seperate-cyano_alone                  62.71511
## Cyanobacteria_moss_seperate-Cyanobacteria_moss_symbiosis 23.99011
##                                                              p adj
## cyano_alone-moss_alone                                   0.7140984
## Cyanobacteria_moss_symbiosis-moss_alone                  0.0937375
## Cyanobacteria_moss_seperate-moss_alone                   0.2614148
## Cyanobacteria_moss_symbiosis-cyano_alone                 0.0071606
## Cyanobacteria_moss_seperate-cyano_alone                  0.0288313
## Cyanobacteria_moss_seperate-Cyanobacteria_moss_symbiosis 0.9476697
## 
## $pH
##                  diff       lwr      upr     p adj
## pH_5.5-pH_3.5 33.6875 10.063467 57.31153 0.0036631
## pH_8.5-pH_3.5 63.4500 39.825967 87.07403 0.0000004
## pH_8.5-pH_5.5 29.7625  6.138467 53.38653 0.0107795

Main question is if cyano_alone and moss_alone grew at different rate than cyano_moss_symbiosis. Graphs show that cyano_moss_symbiosis grows more at high pH, but Tukey HSD suggests that at p-value < 0.05, cyano_moss_symbiosis is only significantly different from cyano_alone and that cyano_moss_separated is different from cyano_alone. This suggests that cyano grows better when in contact or proximity with moss. cyano_moss_separate is nearly significantly different from moss alone (p-value = 0.09 at p-value < 0.1) perhaps indicating that moss benefits marginally from proximity to cyano (hormogonia?), but not as much as cyano benefits from proximity of moss.

2018 Testing ANOVA assumptions

## 
##  Shapiro-Wilk normality test
## 
## data:  tri2018.residuals
## W = 0.9626, p-value = 0.1285

## 
##  Bartlett test of homogeneity of variances
## 
## data:  delta_weight by interaction(condition, pH)
## Bartlett's K-squared = 13.881, df = 11, p-value = 0.2396

## Levene's Test for Homogeneity of Variance (center = median)
##       Df F value Pr(>F)
## group 11  1.4796 0.1821
##       36

1.5 Comparison 2017 & 2018

All Data Summary Stats

ANOVAs with factor(year)

##                 Df Sum Sq Mean Sq F value   Pr(>F)    
## year             1   2465    2465   2.168    0.144    
## condition        3  31575   10525   9.256 1.61e-05 ***
## year:condition   3    974     325   0.285    0.836    
## Residuals      112 127356    1137                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = delta_weight ~ year + condition + year:condition, data = alldata)
## 
## $year
##               diff       lwr      upr     p adj
## 2018-2017 9.252222 -3.197809 21.70225 0.1437031
## 
## $condition
##                                                               diff
## Cyanobacteria_moss_seperate-cyano_alone                   27.09400
## Cyanobacteria_moss_symbiosis-cyano_alone                  42.99933
## moss_alone-cyano_alone                                    11.41100
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate  15.90533
## moss_alone-Cyanobacteria_moss_seperate                   -15.68300
## moss_alone-Cyanobacteria_moss_symbiosis                  -31.58833
##                                                                 lwr
## Cyanobacteria_moss_seperate-cyano_alone                    4.386640
## Cyanobacteria_moss_symbiosis-cyano_alone                  20.291973
## moss_alone-cyano_alone                                   -11.296360
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate  -6.802027
## moss_alone-Cyanobacteria_moss_seperate                   -38.390360
## moss_alone-Cyanobacteria_moss_symbiosis                  -54.295694
##                                                                upr
## Cyanobacteria_moss_seperate-cyano_alone                  49.801360
## Cyanobacteria_moss_symbiosis-cyano_alone                 65.706694
## moss_alone-cyano_alone                                   34.118360
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate 38.612694
## moss_alone-Cyanobacteria_moss_seperate                    7.024360
## moss_alone-Cyanobacteria_moss_symbiosis                  -8.880973
##                                                              p adj
## Cyanobacteria_moss_seperate-cyano_alone                  0.0124514
## Cyanobacteria_moss_symbiosis-cyano_alone                 0.0000163
## moss_alone-cyano_alone                                   0.5581616
## Cyanobacteria_moss_symbiosis-Cyanobacteria_moss_seperate 0.2662823
## moss_alone-Cyanobacteria_moss_seperate                   0.2781954
## moss_alone-Cyanobacteria_moss_symbiosis                  0.0024067

##              Df Sum Sq Mean Sq F value   Pr(>F)    
## year          1   2465    2465   2.365   0.1269    
## pH            2  34698   17349  16.642 4.56e-07 ***
## year:pH       2   6359    3180   3.050   0.0512 .  
## Residuals   114 118847    1043                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

##              Df Sum Sq Mean Sq F value Pr(>F)
## year          1   2465    2465   1.819   0.18
## Residuals   118 159905    1355

pH 3.5 t-tests

## 
##  Welch Two Sample t-test
## 
## data:  ph3.5_cyano_alone_2017$delta_weight and ph3.5_cyano_alone_2018$delta_weight
## t = 1.7477, df = 3.5496, p-value = 0.1645
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -15.26223  60.72890
## sample estimates:
##  mean of x  mean of y 
##   1.383333 -21.350000

## 
##  Welch Two Sample t-test
## 
## data:  ph3.5_moss_alone_2017$delta_weight and ph3.5_moss_alone_2018$delta_weight
## t = -1.1256, df = 3.8116, p-value = 0.3262
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -66.18064  28.53064
## sample estimates:
## mean of x mean of y 
##    13.825    32.650

## 
##  Welch Two Sample t-test
## 
## data:  ph3.5_cmseparate_2017$delta_weight and ph3.5_cmseparate_2018$delta_weight
## t = 0.18795, df = 3.0714, p-value = 0.8626
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -61.40599  69.22266
## sample estimates:
## mean of x mean of y 
##  15.20833  11.30000

## 
##  Welch Two Sample t-test
## 
## data:  ph3.5_cmsymbiosis_2017$delta_weight and ph3.5_cmsymbiosis_2018$delta_weight
## t = 2.2001, df = 6.2444, p-value = 0.06835
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -3.028584 62.568584
## sample estimates:
## mean of x mean of y 
##    44.095    14.325

pH 5.5 t-tests

## 
##  Welch Two Sample t-test
## 
## data:  ph5.5_cyano_alone_2017$delta_weight and ph5.5_cyano_alone_2018$delta_weight
## t = -0.9411, df = 4.3344, p-value = 0.3961
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -57.41747  27.68747
## sample estimates:
## mean of x mean of y 
##     5.635    20.500

## 
##  Welch Two Sample t-test
## 
## data:  ph5.5_moss_alone_2017$delta_weight and ph5.5_moss_alone_2018$delta_weight
## t = -0.2556, df = 7.9984, p-value = 0.8047
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -34.50996  27.62329
## sample estimates:
## mean of x mean of y 
##  32.08167  35.52500

## 
##  Welch Two Sample t-test
## 
## data:  ph5.5_cmseparate_2017$delta_weight and ph5.5_cmseparate_2018$delta_weight
## t = -0.76898, df = 6.7714, p-value = 0.4679
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -74.99429  38.37762
## sample estimates:
## mean of x mean of y 
##  37.71667  56.02500

## 
##  Welch Two Sample t-test
## 
## data:  ph5.5_cmsymbiosis_2017$delta_weight and ph5.5_cmsymbiosis_2018$delta_weight
## t = -0.17764, df = 4.2255, p-value = 0.8672
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -67.37097  59.10764
## sample estimates:
## mean of x mean of y 
##  55.49333  59.62500

pH 8.5 t-tests

## 
##  Welch Two Sample t-test
## 
## data:  ph8.5_cyano_alone_2017$delta_weight and ph8.5_cyano_alone_2018$delta_weight
## t = -1.6909, df = 7.9551, p-value = 0.1295
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -79.11996  12.21330
## sample estimates:
## mean of x mean of y 
##  29.79667  63.25000

## 
##  Welch Two Sample t-test
## 
## data:  ph8.5_moss_alone_2017$delta_weight and ph8.5_moss_alone_2018$delta_weight
## t = -0.65332, df = 7.7814, p-value = 0.5324
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -25.25110  14.14443
## sample estimates:
## mean of x mean of y 
##  24.24667  29.80000

## 
##  Welch Two Sample t-test
## 
## data:  ph8.5_cmseparate_2017$delta_weight and ph8.5_cmseparate_2018$delta_weight
## t = -1.723, df = 6.472, p-value = 0.132
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -93.43573  15.42239
## sample estimates:
## mean of x mean of y 
##  54.04333  93.05000

## 
##  Welch Two Sample t-test
## 
## data:  ph8.5_cmsymbiosis_2017$delta_weight and ph8.5_cmsymbiosis_2018$delta_weight
## t = -2.3212, df = 5.525, p-value = 0.06304
## alternative hypothesis: true difference in means is not equal to 0
## 95 percent confidence interval:
##  -61.988073   2.284739
## sample estimates:
## mean of x mean of y 
##  74.77333 104.62500

2017 & 2018 Comparison Figure

## quartz_off_screen 
##                 2

## quartz_off_screen 
##                 2

2 Metabolite Analysis

2.1 Introduction to design

(in progress)

2.2 metablite change when cyanobacteria pellet is crossfed with moss supernantant

Goal – determine significant changes in metabolites between cyanobacteria and moss using kruskal wallis test.

To look at how suernatant from 1) cyano pellet when incubated with moss supernatant (Ph2-super-Nmusc-SfallaxSupp) compares to 2) moss only supernantant (Ph1-super-Sfallax).

Header- crossfed cyano pellet with moss supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	uptake_up by cyano	0130_NADpositive_11p55	NAD	NAD
0.0138744	eluted_by cyano	0001_4-Pyridoxic_acid_positive_1p32	4-PYRIDOXATE	4-PYRIDOXATE
0.0138744	uptake_up by cyano	0207_UDP-glucose_negative_11p47	URIDINE 5’-DIPHOSPHOGLUCOSE	URIDINE 5’-DIPHOSPHOGLUCOSE / URIDINE 5’-DIPHOSPHOGALACTOSE
0.0138744	uptake_up by cyano	0208_UDP-D-glucose_negative_11p52	URIDINE 5’-DIPHOSPHOGALACTOSE	URIDINE 5’-DIPHOSPHOGLUCOSE / URIDINE 5’-DIPHOSPHOGALACTOSE
0.0179605	eluted_by cyano	0056_2-Hydroxy-4methylthio_butyric_acid_negative_3p66	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID
0.0179605	eluted_by cyano	0223_L-histidine_negative_13p06	L-histidine	L-histidine
0.0201646	eluted_by cyano	0216_L-histidine_negative_11p85	L-HISTIDINE	L-HISTIDINE
0.0201646	uptake_up by cyano	0230_L-ornithine_negative_18p98	L-ORNITHINE	L-ORNITHINE

## [1] "# elluted cyanobacteria metabolites (p.value <= 0.05) is: 65"

## [1] "# taken cyanobacteria metabolites (p.value <= 0.05) is: 79"

2.3 metablite change when moss pellet is crossfed with cyanobacteria supernantant

Goal – determine significant changes in metabolites between cyanobacteria and moss using kruskal wallis test.

Supernatant changes from 1) suernatant from moss pellet when incubated with cyanobacteria supernatant (Ph2-super-Sfallax-NmuscSupp) compared to 2) cyanobacteria pellet only supernantant (Ph1-super-Nmusc).

Header- crossfed moss pellet with cyanobacteria supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	uptake_up by moss	0105_cytidine_negative_6p43	CYTIDINE	CYTIDINE
0.0138744	uptake_up by moss	0174_thiamine_negative_10p28	THIAMINE	THIAMINE
0.0138744	eluted_by moss	0226_ANSERINE_negative_14p47	L-ANSERINE	L-ANSERINE
0.0138744	uptake_up by moss	0056_2-Hydroxy-4methylthio_butyric_acid_negative_3p66	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID
0.0179605	uptake_up by moss	0133_xanthosine_negative_8p42	XANTHOSINE	XANTHOSINE
0.0179605	eluted_by moss	0058_DFructose_positive_7p00	fructose	fructose
0.0201646	eluted_by moss	0130_NADpositive_11p55	NAD	NAD
0.0201646	uptake_up by moss	0150_2-Phospho-D-Glyceric_Acid_positive_12p51	L-2-PHOSPHOGLYCERIC ACID	L-2-PHOSPHOGLYCERIC ACID

## [1] "# elluted moss metabolites (p.value <= 0.05) is: 103"

## [1] "# taken moss metabolites (p.value <= 0.05) is: 44"

2.4 metablite change when fungus pellet is crossfed with cyanobacteria supernantant

Goal – determine significant changes in metabolites between fungus and cyanobacteria using kruskal wallis test.

Supernatant changes from 1) suernatant from fungus pellet when incubated with cyanobacteria supernatant (Ph2-super-Tacro-NmuscSupp) compared to 2) cyanobacteria pellet only supernantant (Ph1-super-Nmusc).

Header- crossfed fungus pellet with cyano supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	uptake_up by fungus	0001_4-Pyridoxic_acid_positive_1p32	4-PYRIDOXATE	4-PYRIDOXATE
0.0138744	uptake_up by fungus	0011_1-Methyladenosine_positive_2p17	1-METHYLADENOSINE	1-METHYLADENOSINE
0.0138744	eluted_by fungus	0218_D-Glycerol_1-phosphate_negative_12p21	SN-GLYCEROL 3-PHOSPHATE	SN-GLYCEROL 3-PHOSPHATE
0.0138744	eluted_by fungus	0142_beta-Glycerophosphoric_acid_positive_11p81	GLYCEROL 2-PHOSPHATE	GLYCEROL 2-PHOSPHATE / SN-GLYCEROL 3-PHOSPHATE
0.0179605	uptake_up by fungus	0082_N-Acetyl-DL-methionine_negative_5p11	N-ACETYL-DL-METHIONINE	N-ACETYL-DL-METHIONINE
0.0179605	uptake_up by fungus	0074_3-METHOXY-L-TYROSINE_positive_8p17	3-METHOXY-L-TYROSINE	3-METHOXY-L-TYROSINE
0.0179605	uptake_up by fungus	0005_1-Hydroxy-2-naphthoic_acid_negative_1p14	1-HYDROXY-2-NAPHTHOATE	1-HYDROXY-2-NAPHTHOATE
0.0201646	uptake_up by fungus	0150_2-Phospho-D-Glyceric_Acid_positive_12p51	L-2-PHOSPHOGLYCERIC ACID	L-2-PHOSPHOGLYCERIC ACID

## [1] "# elluted fungal metabolites (p.value <= 0.05) is: 51"

## [1] "# taken fungal metabolites (p.value <= 0.05) is: 87"

2.5 metablite change when fungus pellet is crossfed with moss supernantant

Goal – determine significant changes in metabolites between fungus and moss using kruskal wallis test.

Supernatant changes from 1) suernatant from fungus pellet when incubated with moss supernatant (Ph2-super-Tacro-SfallaxSupp) compared to 2) moss pellet only supernantant (Ph1-super-Sfallax).

Header- crossfed fungus pellet with moss supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	uptake_up by fungus	0005_dihydrouracil_positive_1p57	5,6-DIHYDROURACIL	5,6-DIHYDROURACIL
0.0138744	eluted_by fungus	0001_4-Pyridoxic_acid_positive_1p32	4-PYRIDOXATE	4-PYRIDOXATE
0.0138744	eluted_by fungus	0174_thiamine_negative_10p28	THIAMINE	THIAMINE
0.0138744	uptake_up by fungus	0095_2deoxyguanosine_negative_6p18	2’-DEOXYGUANOSINE	2’-DEOXYGUANOSINE
0.0138744	uptake_up by fungus	0083_DIPALMITOYL_CEPHALIN_negative_5p15	1,2-DIPALMITOYL-RAC-GLYCERO-3-PHOSPHOETHANOLAMINE	1,2-DIPALMITOYL-RAC-GLYCERO-3-PHOSPHOETHANOLAMINE
0.0179605	eluted_by fungus	0102_Orotic_acid_negative_6p35	OROTATE	OROTATE
0.0179605	eluted_by fungus	0056_2-Hydroxy-4methylthio_butyric_acid_negative_3p66	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID	2-HYDROXY-4-(METHYLTHIO)BUTYRIC ACID
0.0201646	uptake_up by fungus	0011_1-Methyladenosine_positive_2p17	1-METHYLADENOSINE	1-METHYLADENOSINE

## [1] "# elluted fungal metabolites (p.value <= 0.05) is: 118"

## [1] "# taken fungal metabolites (p.value <= 0.05) is: 32"

2.6 metablite change when moss pellet is crossfed with fungus supernantant

Goal – determine significant changes in metabolites between fungus and moss using kruskal wallis test.

Supernatant changes from 1) suernatant from moss pellet when incubated with fungus supernatant (Ph2-super-Sfallax-TacroSupp) compared to 2) fungus pellet only supernantant (Ph1-super-Tacro).

Header- crossfed moss pellet with fungus supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	eluted_by moss	0132_3-Nitro-L-tyrosine_negative_8p33	3-NITRO-L-TYROSINE	3-NITRO-L-TYROSINE
0.0138744	eluted_by moss	0146_AICA_ribonucleotide_positive_12p02	5-AMINOIMIDAZOLE-4-CARBOXAMIDE-1-BETA-D-RIBOFURANOSYL 5’-MONOPHOSPHATE	5-AMINOIMIDAZOLE-4-CARBOXAMIDE-1-BETA-D-RIBOFURANOSYL 5’-MONOPHOSPHATE
0.0138744	eluted_by moss	0105_S-adenosyl-L-homocysteine_positive_10p36	S-(5’-ADENOSYL)-L-HOMOCYSTEINE	S-(5’-ADENOSYL)-L-HOMOCYSTEINE
0.0138744	eluted_by moss	0134_3-Hydroxy-dl-kynurenine_negative_8p42	3-HYDROXYKYNURENINE	3-HYDROXYKYNURENINE
0.0179605	eluted_by moss	0043_2DEOXYURIDINE_negative_2p47	DEOXYURIDINE	DEOXYURIDINE
0.0179605	eluted_by moss	0069_5-Hydroxyindole-3-acetic_acid_positive_7p96	5-HYDROXYINDOLEACETATE	5-HYDROXYINDOLEACETATE
0.0201646	eluted_by moss	0130_NADpositive_11p55	NAD	NAD
0.0201646	uptake_up by moss	0162_N-Acetylneuraminic_Acid_negative_9p99	N-ACETYLNEURAMINATE	N-ACETYLNEURAMINATE

## [1] "# elluted fungal metabolites (p.value <= 0.05) is: 102"

## [1] "# taken fungal metabolites (p.value <= 0.05) is: 21"

2.7 metablite change when cyano pellet is crossfed with fungus supernantant

Supernatant changes from 1) suernatant from cyano pellet when incubated with fungus supernatant (Ph2-super-Nmusc-TacroSupp) compared to 2) fungus pellet only supernantant (Ph1-super-Tacro).

2.7.0.0.0.0.0.1 something is wrong with below code

Header- crossfed moss pellet with fungus supernatant

p.value	met_direction	compound_atlas_ID	label	Identified_metabolite
0.0138744	eluted_by cyano	0133_xanthosine_negative_8p42	XANTHOSINE	XANTHOSINE
0.0138744	eluted_by cyano	0132_3-Nitro-L-tyrosine_negative_8p33	3-NITRO-L-TYROSINE	3-NITRO-L-TYROSINE
0.0138744	eluted_by cyano	0105_S-adenosyl-L-homocysteine_positive_10p36	S-(5’-ADENOSYL)-L-HOMOCYSTEINE	S-(5’-ADENOSYL)-L-HOMOCYSTEINE
0.0138744	eluted_by cyano	0009_5deoxyadenosine_positive_2p03	5’-DEOXYADENOSINE	5’-DEOXYADENOSINE / 2’-DEOXYADENOSINE
0.0138744	eluted_by cyano	0013_2deoxyadenosine_positive_2p42	2’-DEOXYADENOSINE	5’-DEOXYADENOSINE / 2’-DEOXYADENOSINE
0.0138744	eluted_by cyano	0074_3-METHOXY-L-TYROSINE_positive_8p17	3-METHOXY-L-TYROSINE	3-METHOXY-L-TYROSINE
0.0138744	eluted_by cyano	0223_L-histidine_negative_13p06	L-histidine	L-histidine
0.0179605	eluted_by cyano	0069_5-Hydroxyindole-3-acetic_acid_positive_7p96	5-HYDROXYINDOLEACETATE	5-HYDROXYINDOLEACETATE

## [1] "# elluted cyano metabolites (p.value <= 0.05) is: 125"

## [1] "# taken cyano metabolites (p.value <= 0.05) is: 25"